A particularly important design we are now considering involves a tetrad structure, with one affected and one unaffected offspring, in addition to the two parents. This design has been implemented in the Two Sister Study (funded in part by Susan G. Komen for the Cure), which is assessing the joint role of genetic and environmental risk factors in young-onset (under age 50) breast cancer. The discordant sib pair allows estimation of effects of exposures, while the embedded case-parent triad allows detection of haplotypes that confer either protection or risk. The tetrad analyzed together should provide a powerful design for assessing gene-by-environment interaction. We have been working on developing and evaluating methods for use with the tetrad design. The Two Sister Study completed enrollment of nuclear families where one daughter developed breast cancer before age 50 and the other daughter is unaffected. This is described under a separate project. Inherited genotypes, together with tumor characteristics, will need to be explored to investigate factors that predict the clinical course following treatment, and improved statistical methods will also need to be developed in that context. We are undertaking a genome-wide association study based on these data through a contract with the Center for Inherited Disease Research at Johns Hopkins and will be able to explore gene-by-environment effects on risk of young-onset breast cancer and also look at maternally-mediated effects and possible parent-of-origin effects on risk. The genotype data are now here, and with augmentation by imputations carried out at the University of Washington, we now have some 20 million SNPs. The Illumina platform used was the human OmniExpress plus Exome array, and the use of the exome typing will impose the need to develop further methods appropriate for rare alleles. We also are participating in the GAME-ON consortium, which has provided additional SNPs from the newly developed onco-chip. We have begun analyses of these data by developing a risk score based on the SNPs that have previously been replicated in earlier GWAS investigations. In a methodologic extension to our earlier work on case-parent triads, we have developed a robust method to account for parental phenotypes, and applied those methods to our Two Sister Study, in which some 20% of the mothers also had breast cancer. Together with a graduate student from UNC Biostatistics, Alison Wise, we have developed methods for identifying variants on the X chromosome related to risk. We assessed the performance of our new method by applying it to the DbGap data on the birth defect, oral cleft. Our method, the PIX-LRT, makes use of parental information in a robust way in addition to the transmission distortion, and thus makes more efficient use of the data than do existing methods. Alison successfully defended her thesis in April. We are now working on extending the method allow discovery of haplotypes (multiple SNPs close to each other) on the X chromosome and a paper is under preparation. Another project concerns use of the Genetic Algorithm, a stochastic search algorithm, to detect multi-SNP epistatic effects based on transmissions observed from parents to their affected offspring. This ongoing work will be presented at the ASHG meeting in October.